-
Leading Edge
Review
Hematopoiesis: An Evolving Paradigmfor Stem Cell BiologyStuart
H. Orkin1,2,* and Leonard I. Zon1,21Division of
Hematology/Oncology, Children’s Hospital Boston and the Dana Farber
Cancer Institute,
Harvard Stem Cell Institute, Harvard Medical School, Boston, MA
02115, USA2Howard Hughes Medical Institute, Boston, MA 02115,
USA*Correspondence: [email protected]
DOI 10.1016/j.cell.2008.01.025
Establishment and maintenance of the blood system relies on
self-renewing hematopoietic stemcells (HSCs) that normally reside
in small numbers in the bone marrow niche of adult mammals.This
Review describes the developmental origins of HSCs and the
molecular mechanisms that reg-ulate lineage-specific
differentiation. Studies of hematopoiesis provide critical insights
of generalrelevance to other areas of stem cell biology including
the role of cellular interactions in develop-ment and tissue
homeostasis, lineage programming and reprogramming by transcription
factors,and stage- and age-specific differences in cellular
phenotypes.
IntroductionThe blood system serves as a paradigm for
understanding tissue
stem cells, their biology, and involvement in aging, disease,
and
oncogenesis. Because mature blood cells are predominantly
short lived, stem cells are required throughout life to
replenish
multilineage progenitors and the precursors committed to
indi-
vidual hematopoietic lineages. Hematopoietic stem cells
(HSCs) reside as rare cells in the bone marrow in adult
mammals
and sit atop a hierarchy of progenitors that become progres-
sively restricted to several or single lineages (Orkin, 2000).
These
progenitors yield blood precursors devoted to unilineage
differ-
entiation and production of mature blood cells, including
red
blood cells, megakaryocytes, myeloid cells (monocyte/macro-
phage and neutrophil), and lymphocytes. As with all other
stem
cells, HSCs are capable of self-renewal—the production of
addi-
tional HSCs—and differentiation, specifically to all blood
cell
lineages.
HSCs are defined operationally by their capacity to
reconsti-
tute the entire blood system of a recipient. In general,
prepara-
tion of patients for transplantation with donor bone marrow
con-
taining HSCs entails destruction of host bone marrow by
irradiation or by treatment with high-dose cytotoxic drugs,
in
part to provide ‘‘space’’ for donor HSCs within the marrow
mi-
croenvironment (the niche) of the recipient. HSCs can be
pro-
spectively identified by monoclonal antibodies directed to
sur-
face markers, by dye efflux, or on the basis of their
metabolic
properties; HSCs can be separated from more-committed pro-
genitors and other marrow cells by fluorescence-activated
cell
sorting (FACS). With contemporary methods, HSCs may be
highly purified such that as few as one cell may provide
long-
term (>4 months) hematopoietic reconstitution in a
recipient.
Technical considerations regarding the assays for
quantitation
of HSCs and evaluation of their function have recently been
re-
viewed (Purton and Scadden, 2007). Because no ex vivo assays
can replace in vivo transplantation for measuring biological
activity of HSCs, characterizing cell populations based on
the
expression of cell-surface markers cannot be considered
synonymous with determining their function. During stress or
other manipulations (such as in mutant animals), the surface
marker profile of HSCs and their progenitors may be
distorted.
Here, we discuss the developmental origins of the hematopoi-
etic system and the molecular control of self-renewal and
lineage
determination. The process of hematopoiesis is generally
con-
served throughout vertebrate evolution. Manipulation of
animal
models, such as the mouse and zebrafish, has complemented
and greatly extended studies of human hematopoiesis.
Although
not an entirely ideal experimental system, partial
reconstitution of
the blood system of immunodeficient mice (such as NOD/SCID
strains) has been commonly employed to study human hemato-
poiesis. The remarkable regenerative properties of human
HSCs
arebest illustratedby thesuccess of marrow transplantation
inhu-
man patients, a current mainstay of therapy for a variety of
genetic
disorders, acquired states of bone marrow failure, and
cancers.
Emergence of HSCsIn vertebrates, the production of blood stem
cells is accom-
plished by the allocation and specification of distinct
embryonic
cells in a variety of sites that change during development
(Gallo-
way and Zon, 2003) (Figures 1 and 2). In mammals, the
sequen-
tial sites of hematopoiesis include the yolk sac, an area
surrounding the dorsal aorta termed the aorta-gonad meso-
nephros (AGM) region, the fetal liver, and finally the bone
marrow
(Figure 1). Recently, the placenta has been recognized as an
ad-
ditional site that participates during the AGM to fetal liver
period.
The properties of HSCs in each site differ, presumably
reflecting
diverse niches that support HSC expansion and/or
differentia-
tion and intrinsic characteristics of HSCs at each stage. For
in-
stance, HSCs present in the fetal liver are in cycle, whereas
adult
bone marrow HSCs are largely quiescent.
Although there is little dispute regarding where HSCs are
found during development, few topics have polarized
investiga-
tors as much as the origin of HSCs. HSCs are derived from
Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc. 631
mailto:[email protected]
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ventral mesoderm (see Review by C.E. Murry and G. Keller,
page
661 of this issue). The contribution of each hematopoietic
site
(such as the yolk sac and fetal liver) to circulating blood in
the fe-
tus or adult was seemingly answered more than 25 years ago.
Recent studies in mice and zebrafish, however, challenge the
field with divergent views.
Multiple Waves of Hematopoiesis during Development
The initial wave of blood production in the mammalian yolk sac
is
termed ‘‘primitive.’’ The primary function for primitive
hemato-
poiesis is production of red blood cells that facilitate tissue
oxy-
genation as the embryo undergoes rapid growth. The hallmark
of
primitive erythroid cells is expression of embryonic globin
pro-
teins. The primitive hematopoietic system is transient and
rapidly
replaced by adult-type hematopoiesis that is termed
‘‘defini-
tive.’’
In mammals, the next site of hematopoietic potential is the
AGM region. Hematopoietic cells were first detected in the
aorta
of the developing pig more than 80 years ago. Subsequently,
studies of chick-quail chimeras and diploid-triploid Xenopus
em-
bryos demonstrated analogous AGM-like regions. Morphologi-
cal examination revealed that a sheet of lateral mesoderm
mi-
grates medially, touches endoderm, and then forms a single
aorta tube. Clusters of hematopoietic cells subsequently
appear
in the ventral wall. Similarly, an intraembryonic source of
adult
HSCs in mice capable of long-term reconstitution of
irradiated
hosts resides in the AGM region (Muller et al., 1994). At
embry-
onic day 10.5, little HSC activity is detectable, whereas by
day
11 engrafting activity is present.
Figure 1. Developmental Regulation of
Hematopoiesis in the Mouse
(A) Hematopoiesis occurs first in the yolk sac (YS)
blood islands and later at the aorta-gonad meso-
nephros (AGM) region, placenta, and fetal liver
(FL). YS blood islands are visualized by LacZ stain-
ing of transgenic embryo expression GATA-1-
driven LacZ. AGM and FL are stained by LacZ in
Runx1-LacZ knockin mice. (Photos courtesy of
Y. Fujiwara and T. North.)
(B) Hematopoiesis in each location favors the pro-
duction of specific blood lineages. Abbreviations:
ECs, endothelial cells; RBCs, red blood cells; LT-
HSC, long-term hematopoietic stem cell; ST-HSC,
short-term hematopoieticstem cell; CMP, common
myeloid progenitor; CLP, common lymphoid pro-
genitor; MEP, megakaryocyte/erythroid progenitor;
GMP, granulocyte/macrophage progenitor.
(C) Developmental time windows for shifting sites of
hematopoiesis.
Additional hematopoietic activity in the
mouse embryo was detected subse-
quently in other sites, including the umbil-
ical arteries and the allantois in which
hematopoietic and endothelial cells are
colocalized (Inman and Downs, 2007).
Umbilical veins lack hematopoietic po-
tential, suggesting that a hierarchy exists
during definitive hematopoiesis in which
HSCs arise predominantly during artery specification. In
addi-
tion, significant numbers of HSCs are found in the mouse
pla-
centa (Gekas et al., 2005; Ottersbach and Dzierzak, 2005),
nearly
coincident with the appearance of HSCs in the AGM region and
for several days thereafter. Placental HSCs could arise
through
de novo generation or colonization upon circulation, or
both.
The relative contribution of each of the above sites to the
final
pool of adult HSCs remains largely unknown.
Subsequent definitive hematopoiesis involves the
colonization
of the fetal liver, thymus, spleen, and ultimately the bone
marrow.
It is believed that none of these sites is accompanied by de
novo
HSC generation. Rather, their niches support expansion of
pop-
ulations of HSCs that migrate to these new sites. However,
until
very recently (as discussed below), there has been no
evidence
by fate mapping or direct visualization that HSCs from one
site
colonize subsequent sites.
Hemangioblasts and Hemogenic Endothelium
A common origin for blood and vascular cells, the
‘‘hemangio-
blast,’’ was hypothesized a century ago, based largely on the
in-
timate association of these lineages in the blood islands of
the
developing yolk sac. Sharing of markers between blood and
blood vessel cells, and the impairment of both tissues in
mu-
tants, such as the mouse flk1 knockout (Shalaby et al.,
1997)
and zebrafish cloche (Stainier et al., 1995), are consistent
with
a common origin. Clonal studies using in vitro
differentiating
mouse embryonic stem (ES) cells provide the strongest evi-
dence in favor of the existence of hemangioblasts (Choi et
al.,
1998). Furthermore, hemangioblast activity has been detected
632 Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc.
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at the mid-streak stage of gastrulation and during the
neural
plate stage but is extremely transient in vivo (Huber et
al.,
2004). Despite these findings, formal proof of the
hemangioblast
hypothesis requires direct demonstration that a single cell
di-
vides asymmetrically to form blood and vascular derivatives
in vivo.
Clonal analysis in mouse chimeras, however, presents contra-
dictory evidence regarding the existence of the
hemangioblast
(Ueno and Weissman, 2006). Three different, stably marked ES
cells were mixed and coinjected into host blastocysts.
Accord-
ing to the hemangioblast hypothesis, each blood island of
the
yolk sac should be clonally derived. However, in these
experi-
ments more than a single ES cell often contributed to each
blood
island of the chimeric mice. The existence of the
hemangioblast
has also been addressed in zebrafish. A primitive wave of
hema-
topoiesis occurs in a region called the intermediate cell
mass
that contains erythroid cells surrounded by venous
endothelial
cells (see Figure 2). Hematopoietic and endothelial markers
seg-
regate between the 3- to 10-somite period of development. By
this time, there are few, if any, cells that might be considered
he-
mangioblasts based on overlapping blood and blood vessel
gene expression. Alternatively, hemangioblasts could appear
before the 3-somite stage and also exhibit wider
developmental
potential than solely blood and blood vessels. Ventral
mesoder-
mal cells are dedicated specifically to hematopoietic and
endo-
thelial fates. Fate-mapping studies have been performed in
which a caged fluorescent dye is injected into the zebrafish
em-
bryo at the one-cell stage, and then at a later time the
fluorescent
dye is uncaged in single cells using a laser. Individual cells
ap-
pear dedicated to hematopoietic and endothelial lineages at
the 0- to 3-somite stage (Vogeli et al., 2006). However,
other
cell fates may also be present at this early time. Similarly,
smooth
muscle cells can be derived from populations of in vitro
differen-
tiated mouse ES cells exhibiting blood and blood vessel
fates
(Ema et al., 2003; Ema and Rossant, 2003). These studies
sup-
port the existence of hemangioblasts, although it may be
neces-
Figure 2. Hematopoietic Development in
the Zebrafish
(A) Hematopoiesis occurs first in the intermediate
cell mass (ICM) and subsequently in the aorta-go-
nad mesonephros (AGM) region and caudal hema-
topoietic tissue (CHT). Later hematopoietic cells
are found in the kidney as well as in the thymus.
In situ hybridization for GATA-1 at 30 hr (ICM), for
c-myb at 36 hr (AGM), for SCL/tal1 at days 4 and
6.5 (CHT), and for c-myb at day 6 (top view) to dem-
onstrate expression in the kidney marrow and thy-
mus. (Photos courtesy of X. Bai and T. Bowman.)
(B) Developmental time windows for hematopoi-
etic sites in the zebrafish.
sary to redefine the potential of these
cells to include additional lineages (such
as smooth muscle).
Principally based on morphology it has
been proposed that as the AGM forms,
‘‘hemogenic endothelial’’ cells in the ven-
tral wall of the aorta, rather than heman-
gioblasts, bud off HSCs. The program of hemogenic
endothelial
cell development may be regulated differently from that of
pre-
sumptive hemangioblasts, given that the transcription factor
re-
quirements differ. For example, the transcription factor Runx1
is
necessary for blood formation from hemogenic endothelium but
not from yolk sac hemangioblasts (North et al., 1999, 2002).
The
potential to generate hematopoietic, endothelial, and smooth
muscle cells has been attributed to another cell type,
termed
the mesoangioblast, present in the aorta (Cossu and Bianco,
2003). Perhaps, the presumptive mesoangioblast might be a
pre-
cursor of the hemogenic endothelial cell.
Other work has indicated that mesenchymal cell populations
in the subaortic region poke through the aorta and bud off
HSCs (Bertrand et al., 2005). As this occurs, mesenchymal
cells
express endothelial-specific genes and ultimately express
HSC-
associated markers. These observations suggest an
alternative
model in which subaortic mesenchymal cells, which may also
have smooth muscle potential, rather than hemogenic endothe-
lial cells, are the source of future definitive HSCs.
Developmental Relationships between the Yolk Sac
and the AGM
As with mesodermal derivatives, all blood cells in embryonic,
fe-
tal, and adult animals might arise from a small set of cells
during
development. Evidence for and against this notion is present
in
the literature. Fate mapping in the pre-gastrula Xenopus
embryo
with fluorescent dye injected into individual blastomeres of
the
32-cell embryo demonstrated that different blastomeres
contrib-
ute to primitive hematopoiesis and definitive HSC production
(Ciau-Uitz et al., 2000). This finding contradicts the
conclusion
derived from diploid-triploid chimeric frogs that ventral
meso-
derm is the common origin of both primitive and definitive
pop-
ulations (Turpen et al., 1997). Technical aspects of fate
mapping
of the 32-cell embryo have been challenged (Lane and Sheets,
2002).
In situ hybridization and chimera studies in amphibians and
birds suggest that the yolk sac and the AGM are derived
Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc. 633
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independently and arise at different times in development
(Turpen et al., 1997). With short-term culture and
subsequent
transplantation, mouse AGM tissue (isolated one day prior to
the appearance of HSCs in vivo) generates cells with the
capac-
ity for long-term engraftment, whereas mouse yolk sac tissue
does not (Cumano et al., 1996; Medvinsky and Dzierzak,
1996).
The origin of HSCs in the AGM can be traced by Runx1 expres-
sion in the embryonic day 8.5 (E8.5) mouse embryo, just
before
the onset of circulation. Because functional activity of stem
cells
as determined by transplantation into irradiated adults
occurs
much later (at day 11), it is possible that cells of the yolk
sac col-
onize the AGM through the circulation. In fact, HSC-like
activity
of yolk sac cells (as defined by a neonatal transplantation
assay)
(Palis et al., 2001) is detected as early as day 9, although
circu-
lation has started by that time. Conclusive resolution of the
de-
velopmental relationship between cells of the yolk sac and
AGM requires direct visualization of the migration event.
Further-
more, the specific assay used to determine stem cell activity
for
one population of cells (such as immune reconstitution
following
irradiation of adult animals) may not be appropriate for a
different
stem cell population. Distinct host requirements, such as the
use
of neonatal recipients for cells of the yolk sac, may be
necessary.
Some of the intrinsic differences between cell populations,
such
as developmental stage, ease of access, the local niche, and
whether they are dividing, may preclude a host transplant
assay
from detecting engraftment and multilineage reconstitution.
Such questions will plague studies of other tissue stem
cells,
as these stem cells are defined by functional and biological
read-
outs.
Does the Yolk Sac Contain HSCs?
Based on cell fate mapping and transplantation experiments
in
avian and amphibian species, the AGM has been widely viewed
as the principal site for HSC production during vertebrate
devel-
opment. Accordingly, the yolk sac has often been relegated
to
a subservient position, despite older experiments suggesting
that the yolk sac might be the source of adult
hematopoiesis.
Metcalf and Moore cultured E7.5 mouse embryos from which
the yolk sac had been removed (Moore and Metcalf, 1970).
Given
that no hematopoietic cells appeared in the fetal liver
following
several days in culture, they concluded that the yolk sac
was
the major site of adult blood formation for the embryo.
Although
hematopoiesis in the yolk sac is largely primitive in
character,
progenitors within the yolk sac do give rise to definitive type
cells
in hematopoietic colony assays, an observation consistent
with
a yolk sac origin for definitive cells. This view was
supported
by other experiments in which specific donor-derived T cell
pop-
ulations appeared following transplantation of cells of the
yolk
sac into fetuses (Weissman et al., 1978).
In more recent work, Nishikawa and colleagues have also
challenged the dogma that the yolk sac lacks definitive
hemato-
poietic stem cells (Samokhvalov et al., 2007). The fate of
early
embryonic tissues was traced in transgenic mice in which
Runx1 regulatory elements drive expression of hormonally
acti-
vated Cre recombinase. Administration of tamoxifen to
pregnant
female mice at a particular developmental window permits the
fate of cells expressing Runx1 (visualized by activation of
a Flox-LacZ allele) to be assessed. Treatment of embryos at
E7.5 led to prominent marking of fetal liver cells and adult
hema-
634 Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc.
topoietic cells. As the yolk sac is the only hematopoietic site
at
E7.5 and the only tissue known to express Runx1 at E7.5,
these
findings suggest that the yolk sac contains definitive HSCs
(or
cells that may give rise to HSCs). These experiments were
inter-
preted to support the yolk sac as a site of HSC formation prior
to
the AGM, although consensus in the field is far from
unanimous
(DeWitt, 2007). Diploid-triploid transplants in frogs reveal
that
�20% of adult blood in some animals is derived from the
ventralblood island (the equivalent to the yolk sac), providing
indepen-
dent evidence that adult hematopoiesis may arise from the
yolk
sac region. Despite this finding, it is also clear that the
analogous
AGM region in Xenopus is the predominant contributor to
adult
hematopoiesis. The precise origin of HSCs in the adult
remains
a topic for further debate and study.
In Vivo Fate Mapping of Migrating Cells
Presumptive HSCs in the zebrafish express the transcription
fac-
tors c-myb and Runx1 (see Figure 2). Caged fluorescein dye
fate
mapping of AGM cells has revealed a new hematopoietic
region,
the caudal hematopoietic tissue. Laser uncaging is targeted
to
a region of cells in which transgenic expression of green
fluores-
cent protein (GFP) driven by an HSC-specific promoter marks
HSCs in the AGM (Ferkowicz et al., 2003). This approach
ensures
that laser uncaging occurs specifically within HSCs.
Multiple
cells are uncaged and their fate is followed (Jin et al., 2007;
Mur-
ayama et al., 2006). Uncaged cells of the AGM region that
ex-
press CD41 (a surface marker of early HSCs) or c-myb appear
later as fluorescent cell populations in the caudal
hematopoietic
tissue. The larval and adult site of hematopoiesis in the
zebrafish
is the kidney. Later on in the fate map experiments, the larval
kid-
ney becomes fluorescent, demonstrating that cells of the
caudal
hematopoietic tissue colonize the kidney. In addition,
fluores-
cence is detected in the thymus. Recent evidence suggests
di-
rect population of thymic primordia through tissue planes, a
find-
ing consistent with earlier experiments in birds showing
migration
of progenitors to the thymus along the thoracic duct. Thus,
pop-
ulation of the thymus may occur through circulation and
direct
migration through tissues. Alternatively, the caudal
hematopoi-
etic tissue may represent a site similar to the placenta or
fetal liver
prior to the onset of definitive hematopoiesis in the
kidney.
It is generally stated that HSCs of the fetal liver circulate to
the
adult bone marrow and, hence, are the source of adult
hemato-
poiesis in birds and mammals (see Review by D.J. Laird et
al.,
page 612 of this issue). In contrast, developmental studies
reveal that the fetal liver and marrow are seeded at similar
times
during development (Delassus and Cumano, 1996). Direct
track-
ing of cellular migration is required to distinguish these
possibil-
ities.
Pathways Involved in the Emergence of HSCs
The AGM has been characterized largely by morphology and
functional assays, but the pathways involved in HSC
generation
remain incompletely defined. Studies of chick embryos demon-
strate that endoderm has a prominent role and secretes
inducing
factors. Somitic mesoderm also contributes to the dorsal
aspect
of the aorta, and the addition of factors—such as VEGF,
TGF-b,
and FGF—to the somitic mesoderm leads to induction of hema-
topoietic tissue. In contrast, TGF-a and EGF suppressed
forma-
tion of hematopoietic cells (Pardanaud and Dieterlen-Lievre,
1999).
-
Signaling pathways that regulate the induction of the AGM
have recently been uncovered in mouse and zebrafish. Notch 1
is required for artery identity and aortic HSC production
(Kumano
et al., 2003). In the zebrafish mutant mindbomb that lacks
Notch
signaling, Runx1 overexpression rescues HSC production
(Burns et al., 2005). Similarly, a Notch1 mutant is rescued
by
Runx1 overexpression, suggesting that Runx1 lies downstream
or parallel to Notch signaling. Other pathways participate in
the
process including CoupTF-II (Pereira et al., 1999), as well
as
the CDX-HOX pathway (Davidson et al., 2003).
The Wnt/b-catenin and Notch-Delta signaling pathways influ-
ence the function of adult HSCs. Treatment of purified HSCs
with
Wnt3a protein leads to a modest increase in engrafting cells
(Reya et al., 2003). Whereas a pulse of Wnt signaling
appears
to induce HSCs, constitutive Wnt activation by stabilized
b-cat-
enin leads to anemia, possibly by stem cell exhaustion as a
con-
sequence of prolonged Wnt signaling (Kirstetter et al.,
2006;
Scheller et al., 2006). Wnt signaling may be dispensable for
adult
HSC homeostasis, given that conditional knockout of b- or
g-catenin in hematopoietic cells fails to affect HSC number
or
engraftment potential (Cobas et al., 2004; Scheller et al.,
2006).
Stimulation of the Notch pathway also increases HSC activity
and appears to be required for the increased self-renewal
upon Wnt activation (Duncan et al., 2005). In addition to
the
Wnt and Notch pathways, new growth factors such as angio-
poietin-like proteins appear capable of supporting ex vivo
expansion of HSCs (Zhang et al., 2006).
A chemical genetic screen has recently revealed a role for
the
prostaglandin pathway in the production of HSCs in the
zebra-
fish. Treatment of embryos with prostaglandin E2 (PGE2) aug-
ments stem cell production (North et al., 2007), most likely
through the EP4 receptor, a G-coupled receptor specifically
ex-
pressed in the aorta region and activated by PGE2
(Villablanca
et al., 2007). Prostaglandins also affect the homeostasis of
defin-
itive adult hematopoiesis, as shown by irradiation recovery
as-
says, 5-fluorouracil stimulation assays, and long-term
hemato-
poietic reconstitution. Thus, the emergence of HSCs in the
aorta involves the prostaglandin pathway and the Notch-Runx
pathways, which appear to be independent based on genetic
relationships.
The hematopoietic system of the Drosophila embryo gener-
ates myeloid-like cells critical for tissue remodeling and
engulf-
ment and phagocytosis of dead cells. The emergence of sites
of hematopoiesis during embryogenesis is remarkably similar
to that of vertebrates. Drosophila progenitors are also
formed
adjacent to the circulatory system. Hematopoietic
progenitors
bud off from head mesoderm. These myeloid cells are
transient
and ultimately replaced by cells that bud off near the heart
re-
gion and the great vessel. Vascular endothelial growth
factor
(VEGF) ligands are required for derivation of adult
hematopoi-
etic cells, as well as for attracting myeloid cells at specific
sites
(Cho et al., 2002). Genetic analysis demonstrates that
specific
signaling pathways, such as Notch, are required for the
deriva-
tion of the lymph gland and a hemangioblast-like cell
population
(Mandal et al., 2004). Recent studies demonstrate that the
lymph gland of the third instar larva of the fruit fly is
patterned
and contains a signaling center that expresses Hedgehog li-
gand (Krzemien et al., 2007; Mandal et al., 2007). Hedgehog
co-
operates with Notch ligands expressed in these regions to
form
a stem cell niche and regulates the cycling of hematopoietic
progenitors. The search is underway for a similar signaling
cen-
ter in vertebrates. Hedgehog is also required for AGM
hemato-
poiesis in the zebrafish (Gering and Patient, 2005). More
re-
cently, studies of human embryonic stem cells have indicated
that factors such as hedgehog and bone morphogenetic
protein (BMP) promote blood production during in vitro
differ-
entiation.
NichesStem cells depend on their microenvironment, the niche,
for reg-
ulation of self-renewal and differentiation. Studies of
Drosophila
testes and ovarian stem cells have led to formulation of
concepts
that may be applicable to the niche in other tissues (Decotto
and
Spradling, 2005; see also the Review by S.J. Morrison and
A.C.
Spradling, page 598 of this issue). For instance, in the
ovary,
a hub cell directly binds to a stem cell and regulates its
self-re-
newal and differentiation, in part though BMP signaling (see
Mini-
review by R.M. Cinalli et al., page 559 of this issue). In the
testis,
an apical hub cell expresses the ligand Upd, an activator of
the
JAK-STAT signaling pathway in adjacent germ cells to control
their self-renewal. By analogy to the Drosophila reproductive
or-
gans, investigators have sought an equivalent of the hub cell
for
the HSC.
As the site of hematopoiesis changes during vertebrate
devel-
opment, the nature of the stem cell niche must also change.
The
adult bone marrow niche (depicted in Figure 3) has received
most attention. Mutant mice in which the BMP pathway is dis-
rupted have increased numbers of osteoblasts and HSCs (Calvi
et al., 2003; Zhang et al., 2003). These findings suggest that
os-
teoblasts may represent a critical component of the bone
mar-
row niche for HSCs. As assessed by intravital microscopy,
HSCs appear to reside in the periosteal region of calverium
mar-
row (Sipkins et al., 2005). Transplanted GFP-marked or LacZ-
marked HSCs appear to lodge adjacent to osteoblasts. Many
factors, including ligands for Notch receptors and
N-cadherin,
are liberated by osteoblasts, although the contribution of
these
to adult hematopoiesis remains to be established. The role
of
N-cadherin as a mediator of interactions with osteoblasts
(Zhang
et al., 2003), as well as the prominence of osteoblasts for
HSC
adherence, have been challenged (Kiel et al., 2007). Recent
find-
ings suggest that HSCs are maintained in a quiescent state
through interaction with thrombopoietin-producing
osteoblasts
(Yoshihara et al., 2007). The association of HSCs with
osteo-
blasts is countered by other studies that place HSCs
adjacent
to vascular cells. The chemokine CXCL12 regulates the migra-
tion of HSCs to the vascular cells (now called the vascular
niche)
(Kiel and Morrison, 2006). Taken together, these findings
sug-
gest that HSCs reside in various sites within the marrow and
that their function might depend on their precise
localization.
Much of the existing debate may be semantic, however, if the
os-
teoblastic and vascular niches are intertwined and not
physically
separate. Alternatively, HSCs may truly reside in distinct
subre-
gions, which may endow them with different activities.
Cellular
dynamics within the niche are relevant to clinical marrow
trans-
plantation. For example, recent findings suggest that
antibody-
mediated clearance of host HSCs facilitates occupancy of the
Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc. 635
-
niche and transplantation by exogenous HSCs (Czechowicz
et al., 2007).
How niches modulate self-renewal is a challenge for future
studies. The generation of premalignant myeloproliferative
syn-
dromes in mice with abnormal niches underscores the need for
precise control in vivo (Perry and Li, 2007). Remarkably,
the
site of hematopoiesis is not conserved in vertebrate
evolution.
For instance, the site of adult hematopoiesis is the kidney
in
fish. The frog forms adult blood in the liver, and birds and
mam-
mals form blood in the marrow. In the frog Rana temporaria,
the
site of hematopoiesis switches between the liver and bone
mar-
row depending on the season (Maslova and Tavrovskaia, 1993).
Little is known regarding the nature of the niche for
embryonic
hematopoietic sites. Are diverse properties attributed to
embry-
onic, fetal, and adult HSCs due to differences in their
respective
niches? To what extent are factors shared among different
de-
velopmental or anatomical niches?
Transcription Factors in Hematopoietic DevelopmentAs intrinsic
determinants of cellular phenotype, transcription fac-
tors provide an entry point for unraveling how HSCs develop
dur-
ing embryogenesis and how lineage-restricted differentiation
is
programmed (Orkin, 2000). Here we focus on principles and
con-
cepts that have emerged and consider how these may inform
other organ/tissue systems. Recent reviews provide
additional
discussion of transcription factors in different hematopoietic
lin-
eages (Nutt and Kee, 2007; Iwasaki and Akashi, 2007; Kim and
Bresnick, 2007; Rothenberg, 2007). Insights into the
functions
of the critical transcription factors have rested
predominantly
on findings from either conventional or conditional gene
knock-
Figure 3. Stem Cell Niche in the Adult Bone Marrow
HSCs are found adjacent to osteoblasts that are under the
regulation of bone
morphogenetic protein (BMP) (the osteobast niche). HSCs are also
found ad-
jacent to blood vessels (the vascular niche). The chemokine
CXCL12 regulates
the migration of HSCs from the circulation to the bone marrow.
The osteoblast
and vascular niches in vivo lie in close proximity or may be
interdigitated. The
marrow space also contains stromal cells that support
hematopoiesis includ-
ing the production of cytokines, such as c-Kit ligand, that
stimulate stem cells
and progenitors. Cytokines, including interleukins,
thrombopoietin (Tpo), and
erythropoietin (Epo), also influence progenitor function and
survival.
636 Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc.
outs in mice and from forced expression experiments, all
com-
plemented by developmental studies in other model organisms
(e.g., zebrafish, chicken, Drosophila, Xenopus). The
transcription
factors that are critical for hematopoiesis encompass virtually
all
classes of DNA-binding proteins, rather than favoring a
specific
family. A remarkable feature of transcription factors in the
hema-
topoietic system is that the majority are involved in
chromosomal
translocations or with somatic mutations in human hematopoi-
etic malignancies. Furthermore, experimental manipulation of
the genes for such factors in mice often promotes
malignancy.
Hematopoietic cell fate is intertwined with the origins of
leuke-
mias. Requirements for several transcription factors, as
estab-
lished through conventional gene targeting, are summarized
in
Figure 4 (see also the SnapShot by S.H. Orkin and L.I. Zon,
page 631 of this issue).
For discussion purposes, one may distinguish between fac-
tors required for HSC formation or function and those
employed
in lineage-specific differentiation. Among the ‘‘HSC
transcription
factors’’ are MLL (for mixed lineage-leukemia gene), Runx1,
TEL/
ETV6, SCL/tal1, and LMO2, whose genes account in toto for
the
majority of known leukemia-associated translocations in pa-
tients. In these instances, the translocations either
deregulate
expression of the locus, as in the case of SCL/tal1 and LMO2
in T cell acute leukemias, or generate chimeric fusion
proteins,
as in myeloid and lymphoid leukemias associated with MLL,
Runx1, and TEL/ETV6. The above distinction is arbitrary in
that
all of these HSC transcription factors also serve roles later
within
differentiation of individual blood lineages, and conversely,
fac-
tors that appear to have more lineage-restricted roles (such
as
PU.1, Gfi-1, C/EBPa) act within HSCs. The redeployment of
tran-
scription factors at different stages of blood cell development
is
reflected in part in the dynamic patterns of expression of key
reg-
ulators and complicates analysis of in vivo requirements.
Both
temporal- and lineage-restricted conditional inactivation are
of-
ten needed to reveal a meaningful phenotype. These circum-
stances reflect parsimony with regard to the repertoire of
factors
required to achieve complex gene regulation and
differentiation.
Factors Essential for Formation of HSCs
The transcription factors required to program mesoderm
toward
a hematopoietic fate are of special interest. The
basic-helix-
loop-helix (bHLH) factor SCL/tal-1 and its associated
protein
partner, the Lim-domain containing LMO2, are individually
es-
sential for development of both the primitive and definitive
(or
adult) hematopoietic systems (Kim and Bresnick, 2007). In
their
absence, no blood cells are generated. Within the primitive
sys-
tem at the yolk sac stage, these factors are thought to
function
within the hemangioblast to specify a blood rather than a
vascular
fate. The genes encoding the SET-domain containing histone
methyltransferase MLL and runt-domain Runx1 proteins are es-
sential for generation of HSCs within the AGM (and possibly
at
other sites) (Orkin, 2000). In the absence of Runx1, no
hemato-
poietic clusters (representing presumptive HSCs) form in the
dorsal aorta in mice. As noted above, in zebrafish Runx1
lies
downstream of Notch signaling, which is required for the
induc-
tion of hematopoiesis. In addition, BMP signaling restricts
hem-
ato-vascular development of lateral mesoderm, possibly
acting
through a pathway involving LMO2 and presumably GATA-2
(Burns et al., 2005). MLL, like its Drosophila counterpart
trithorax,
-
functions in maintenance of, but not initiation of, HOX gene
ex-
pression. MLL also appears to lie upstream of HOXB4 (and
pre-
sumably other HOX genes) in HSC specification. Leukemias in
which MLL function is perturbed due to chromosomal
transloca-
tions may arise in part as a secondary consequence of
changes
in HOX gene expression. Recently, it has been demonstrated
that expression of an MLL fusion gene (MLL-AF9) in granulo-
cyte/macrophage progenitors (GMPs) induces a ‘‘HSC stem
cell-like’’ signature that includes various HOX genes
(Krivtsov
et al., 2006). The acquisition of a stem cell signature by
leukemic
GMPs may contribute to self-renewal of leukemia stem cells.
Study of a zebrafish mutant defective for blood formation
iden-
tified the caudal-related factor Cdx4 as an inducer of blood
specification (Davidson et al., 2003). Indeed, Cdx4-deficient
em-
bryos are rescued by expression of various homeobox genes
(e.g., HoxA9) but not by SCL/tal1 (Yan et al., 2006).
Activation
of Cdx4 expression in mouse ES cells alters the pattern of
HOX gene expression, augments in vitro blood formation, and
cooperates with HOXB4 in the generation of long-term
reconsti-
tuting hematopoietic progenitors (Wang et al., 2005). Recent
evidence indicates that the pathway to Cdx4 in the zebrafish
is
initiated by the TATA-box binding protein-related factor 3
(Trf3)
(Hart et al., 2007).
Temporal and Stage-Specific Requirements
for Hematopoietic Regulators
Within the definitive hematopoietic system, fetal liver and
bone marrow HSCs differ in many properties, including cell-
surface markers, developmental potential, and cell-cycle
status.
Figure 4. Requirements of Transcription Factors in
Hematopoiesis
The stages at which hematopoietic development is blocked in the
absence of a given transcription factor, as determined through
conventional gene knockouts,
are indicated by red bars. The factors depicted in black have
been associated with oncogenesis. Those factors in light font have
not yet been found translocated
or mutated in human/mouse hematologic malignancies.
Abbreviations: LT-HSC, long-term hematopoietic stem cell; ST-HSC,
short-term hematopoietic stem
cell; CMP, common myeloid progenitor; CLP, common lymphoid
progenitor; MEP, megakaryocyte/erythroid progenitor; GMP,
granulocyte/macrophage progen-
itor; RBCs, red blood cells.
Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc. 637
-
Transplantation experiments in mice suggest that some
charac-
teristics that distinguish fetal liver and adult HSCs are
intrinsically
regulated (Bowie et al., 2007). Furthermore, HSCs in older
age
mice exhibit different self-renewal and gene expression
patterns
than those from younger animals (see Review by D. Rossi et
al.,
page 681 of this issue). How the distinctive properties of HSCs
at
different developmental stages are programmed is of
particular
interest in correlating biological read-outs with molecular
deter-
minants. Recently, the HMG-box containing factor Sox17,
which
is also critical to endoderm specification, has been identified
as
critical for generation of fetal, but not adult, HSCs (Kim et
al.,
2007). ‘‘Geriatric’’ HSCs are less efficient at homing to and
en-
grafting in the bone marrow, possibly linked to their
increased
cycling frequency. In addition, the differentiation potential
of
older HSCs is biased toward myeloid versus lymphoid lineages
(Sudo et al., 2000). Several changes in gene expression of
old
versus young HSCs have been described, including increased
expression of leukemia-associated genes and decreased ex-
pression of genes contributing to DNA damage repair, genomic
integrity, and chromatin remodeling (Rossi et al., 2005;
Nijnik
et al., 2007). Some of these properties are posited to
predispose
older HSCs to myeloid leukemias (see Review by D. Rossi et
al.).
Moreover, the transcription factors required for
specification
and formation of HSCs may not be required continuously for
the subsequent survival or self-renewal of HSCs. Although
SCL/tal1 is an obligate factor for hematopoietic fate
specification
during development, conditional inactivation in adult HSCs
has
surprisingly little consequence on maintenance or
self-renewal
of HSCs and multipotent progenitors (Mikkola et al., 2003).
Un-
der these circumstances, the role of this factor in maturation
of
erythroid and megakaryocytic cells is revealed. Similarly,
inacti-
vation of Runx1 in adult HSCs does not ablate HSC properties
but instead perturbs differentiation of specific lineages
(mega-
karyocytes, lymphocytes) (Ichikawa et al., 2004). Such
observa-
tions point to differences in the transcription factor
composition
of emerging HSCs and adult HSCs and suggest that the pheno-
type of HSCs is quite stable.
Multilineage Gene Expression in HSCs
Generally, the expression of the lineage-affiliated
transcription
factors can be readily reconciled with the simple hierarchy
dia-
grams of hematopoiesis (see Figures 1 and 4). For instance,
GATA-1 is highly expressed in megakaryocytic/erythroid pro-
genitors (called MEPs) that give rise to megakaryocyte and
red blood cell precursors, whereas a ‘‘myeloid factor,’’
such
as C/EBPa, is present in GMPs. Indeed, in committed progeni-
tors and precursors one can conveniently match cell-surface
phenotypes (defined by monoclonal antibodies) and the subset
of hematopoietic transcription factors expressed in these
cells.
However, this relationship breaks down at earlier stages in
the
hierarchy. A simple one-to-one correspondence of lineage-re-
stricted transcription factors and progenitors is challenged
by
findings that earlier multipotential progenitors and HSCs
ex-
press markers of disparate lineages even within single cells,
al-
beit generally at low levels (Orkin, 2003). This phenomenon,
termed lineage priming, suggests that the fate of these
imma-
ture cells is not sealed and that lineage selection is largely a
pro-
cess in which alternative possibilities are extinguished
rather
than one in which new programs are imposed on an otherwise
blank slate.
Lineage priming may be an efficient means by which chroma-
tin invested in important hematopoietic programs is
maintained
in an available or open configuration in HSCs. Transient
repres-
sion of alternative fates, followed by more permanent
silencing,
maintains the inherent plasticity of multipotential
progenitors.
Moreover, the coexistence of transcription factors
representing
different lineages within a common cell (the HSC or immature
progenitor) offers the potential for immediate ‘‘crosstalk’’
be-
tween different fates at the molecular level (see below).
Recently, by FACS sorting of cells initiating expression
GATA-
1 or PU.1, it has been demonstrated that short-term
repopulating
HSCs may be further subdivided into those committed to mye-
loerythroid and myelolymphoid lineages (Arinobu et al.,
2007).
As these findings illustrate, continued fractionation of HSC
or
progenitor populations reveals increasing diversity in the
choice
of lineage. Thus, the schematic lineage diagrams that are
gener-
ally presented cannot be taken literally but rather as guides to
the
options available to progenitors. The extent to which HSCs
ex-
hibit developmental potential beyond hematopoiesis remains
controversial (Graf, 2002). Experiments purportedly
demonstrat-
ing ‘‘plasticity’’ through transplantation of marrow cells to
recip-
ient mice are plagued by possible cell fusion of
differentiated
hematopoietic cells with host cells and by inadequate
character-
ization of input populations.
Mechanisms of Action for Principal Hematopoietic
Regulators
The requirements and functions of the principal
transcriptional
regulators are context dependent (Orkin, 2000). The key
line-
age-restricted factors are endowed with the complementary
tasks of promoting their own lineage differentiation while
simul-
taneously acting against factors favoring other choices (Figure
5).
Combining positive and antagonistic roles in the major
regulators
provides an efficient means for resolving and reinforcing
lineage
Figure 5. Transcription Factor Antagonism in Lineage
Determina-
tion
Examples of antagonism are depicted in red. The transcription
factors present
in the mature precursors following choice of specific lineage
are shown at the
bottom in black. Abbreviations: CMP, common myeloid progenitor;
MEP,
megakaryocyte/erythroid progenitor; GMP, granulocyte/macrophage
progen-
itor; RBCs, red blood cells.
638 Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc.
-
choices. Numerous examples of this principle of lineage pro-
gramming have been described. GATA-1 and PU.1 promote ery-
throid/megakaryocytic/eosinophil and myeloid
differentiation,
respectively. The proteins physically interact and
antagonize
each other’s actions. In vivo confirmation of the
complementary
roles of GATA-1 and PU.1 has been shown in zebrafish.
Inhibi-
tion of GATA-1 expression by morpholinos shifts
hematopoietic
progenitors to a myeloid fate, whereas the converse occurs
upon
inhibition of PU.1 expression (Galloway et al., 2005; Rhodes
et al., 2005). Other examples of direct antagonism by
hematopoi-
etic transcription factors include the relative relationships of
C/
EBP and FOG1 with respect to eosinophil and multipotential
cell fates (Querfurth et al., 2000), EKLF and Fli-1 for
erythroid
and megakaryocytic choice (Starck et al., 2003), GATA-3 and
T-bet for TH1 and TH2 cells (Usui et al., 2006), and Gfi1
and
PU.1 for neutrophil versus monocyte outcomes (Dahl et al.,
2007). In the absence of Gfi1 in mice, neutrophil precurors
fail
to mature and also incompletely silence monocyte/macrophage
gene expression (Hock et al., 2003).
The critical contribution of repression to lineage selection is
il-
lustrated by loss-of-function studies of Pax5. Proper B cell
devel-
opment requires Pax5 (Nutt and Kee, 2007). In its absence,
proB
cells assume a multipotential phenotype and differentiate
(under
appropriate growth factor conditions) to T-, NK-, or
dendritic
cells, macrophages, neutrophils, or erythroid precursors.
Re-
pression of critical growth factor receptors, for example
macro-
phage-colony-stimulating factor (M-CSF) receptor, restricts
line-
age choice during normal development. In effect, Pax5
commits
progenitors to a B cell fate, while other B cell transcription
fac-
tors, such as E2A and EBP, specify lineage-appropriate gene
activation.
In T-lymphoid development, Notch signaling serves a similar
but more focused role as it functions as a commitment
factor,
principally by repressing factors, such as PU.1, associated
with other outcomes. Although GATA-3 has been viewed as
a T cell-specific transcription factor, recent work indicates
that
it functions only in the context of Notch signaling to promote
T
cell development (Rothenberg, 2007). As recently stated,
lineage
programming in the T cell lineage is more the consequence of
‘‘negotiation’’ rather than instruction.
Mechanisms of Lineage Programming
The context-dependent action and direct antagonism between
key transcriptional regulators are best accommodated by
models in which factors interact directly within protein
com-
plexes (Orkin, 2000). In this regard, the GATA factors are
illustra-
tive (Kim and Bresnick, 2007). A protein complex in
erythroid
cells comprised of GATA-1 (or its close relative, GATA-2),
LMO2 and its partner Ldb1, and SCL/tal1 and its
heterodimeric
partner E2A recognizes a consensus GATA-E-box DNA motif.
Knockout of either LMO2 or SCL/tal1 leads to the absence of
any hematopoietic progenitors in the early embryo. The
identical
phenotypes are consistent with the action of these proteins
within a single complex that is required prior to any
commitment
to erythroid differentiation. Indeed, forced expression of
GATA-
1/2, SCL, and LMO2 converts Xenopus mesoderm efficiently
to a hematopoietic fate. Remarkably, although the GATA/SCL/
LMO2/Lbd1 complex recognizes a composite DNA-binding
site, later studies demonstrated that the DNA-binding
activity
of SCL (in a heterodimer with E2A) is dispensable for
hematopoi-
etic specification but required for full erythroid and
megakaryo-
cytic cell maturation (Porcher et al., 1999). The recruitment
of
DNA-binding-defective SCL to the protein complex accounts
for in vivo function despite its inability to bind to DNA.
GATA factors form alternative protein complexes with a spe-
cific cofactor known as FOG (for friend of GATA) (Kim and
Bres-
nick, 2007). Targeted mutation in mice has demonstrated the
es-
sential role of the GATA/FOG interaction for erythroid and
megakaryocytic development. The GATA-1 (or GATA-2)/FOG1
complex in both erythroid and megakaryocytic lineages is
phys-
ically associated with the NuRD chromatin remodeling complex
via an NuRD binding motif at the N terminus of FOG1. The
inter-
action of FOG1 with GATA factors mediates transcription
repres-
sion and also may facilitate accessibility of the GATA factor to
its
DNA-binding motif in chromatin. GATA/SCL/LMO2/Ldb1 and
GATA/FOG1 complexes appear to be mutually exclusive.
Other connections between hematopoietic factors and chro-
matin-associated proteins amplify this theme. Ikaros
proteins
also associate with the NuRD complex and participate in
repres-
sion and the formation of heterochromatin (Kim et al., 1999).
The
erythroid factor EKLF interacts with Brg1, a critical component
of
the Swi/Snf ATP-dependent chromatin remodeling complex
(Brown et al., 2002). Consistent with the relevance of this
rela-
tionship to biological function, a Brg1 mutant protein, which
re-
tains ATPase activity and assembles into the Swi/Snf
complex,
fails to generate DNase I hypersensitivity and leads to
embryonic
lethality due to failure to activate normal b-globin
expression
(Bultman et al., 2005). Furthermore, another erythroid
factor
NF-E2 associates with the MLL2 complex, which is responsible
for H3K4 histone methylation (Demers et al., 2007). In
addition,
the zinc-finger repressor Gfi protein recruits a
CoREST/lysine
demethylase (LSD1) complex to target genes in erythroid,
mega-
karyocytic, and myeloid cells to control maturation of these
line-
ages (Saleque et al., 2007). Further elucidation of the ways
in
which hematopoietic transcription factors interact and
function
with chromatin-associated factors in HSCs and individual
line-
ages is an on-going challenge for the field.
The involvement of multiprotein complexes in the action of
he-
matopoietic transcription factors predicts that relative
concen-
trations of particular factors should influence the choice of
line-
age. Protein complexes constitute a convenient platform for
direct competition, as well as for cooperative action.
Consider-
able data argue for concentration-dependent effects in
lineage
choice and differentiation. Experiments in zebrafish cited
above
in which the pattern of erythroid versus myeloid development
is
affected by inhibition of expression of GATA-1 or PU.1
(Galloway
et al., 2005; Rhodes et al., 2005) are consistent with this
model.
The relative action of GATA-1 and PU.1 fits a simple
quantitative
model that predicts a metastable undifferentiated progenitor
state when both proteins are present at low levels and
differen-
tiation to one of two alternatives when one of the proteins is
pres-
ent at higher levels (Roeder and Glauche, 2006). Taking
advan-
tage of PU.1-null fetal liver progenitors, Singh and
colleagues
have proposed that high-level PU.1 expression directs macro-
phage differentiation, whereas low-level expression promotes
B cell formation (DeKoter and Singh, 2000). Subsequently,
they proceeded to show that low-level PU.1-expressing
Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc. 639
-
bi-phenotypic (macrophage/neutrophil) cells are relatively
sta-
ble, and increased PU.1 levels promote macrophage
differentia-
tion by influencing a circuit of counterantagonistic
repressors,
Egr-1/2/Nab-2 and Gfi1 (Laslo et al., 2006). A mathematical
model, similar to that described above for GATA-1 and PU.1,
has been derived to account for these observed effects in
the
two myeloid lineages.
MicroRNAs (miRNAs) provide an additional level of control
be-
yond the transcription factors (Shivdasani, 2006) (see
Minireview
by B. Stadler and H. Ruohola-Baker, page 563 of this issue).
On-
going studies of the involvement of miRNAs in hematopoiesis
will
reveal their roles in lineage decisions, stem cell to
progenitor
transitions, niche control, and cell function. The transcription
of
blood cell-specific miRNAs is likely to be driven by the
com-
plexes discussed above, providing a complex regulatory net-
work. Several miRNAs are highly expressed in specific
hemato-
poietic lineages and manipulation of their levels has been
correlated with changes in cellular properties or
differentiation
(Chen et al., 2004). For example, miR-150 impinges on B cell
dif-
ferentiation by targeting c-myb mRNA (Xiao et al., 2007),
whereas miR-155 is required for T helper cell generation and
ger-
minal center activity (Rodriguez et al., 2007; Thai et al.,
2007).
Moreover, conditional inactivation of the gene encoding
Dicer,
an essential component in the processing of pre-miRNAs to
miRNAs, leads to multiple defects in T-lineage lymphopoiesis
(Muljo et al., 2005; Cobb et al., 2006). Unpublished
experiments
indicate that Dicer is also required in the setting of bone
marrow
transplantation for radioprotection of lethally irradiated
recipient
mice (cited in Martinez and Busslinger, 2007).
Lineage Reprogramming
Cellular differentiation was once considered unidirectional,
that
is, once progenitors have committed to a particular linear
path-
way their fate is sealed. Accumulating evidence in the
hemato-
poietic system and in other systems (see Review by R.
Jaenisch
and R. Young, page 567 of this issue) dispels this notion and
pro-
vides a strong foundation for cellular reprogramming.
Indeed,
cells of one hematopoietic lineage can be converted to
another
through the forced expression of carefully chosen
transcription
factors (Figure 6). Knowledge of the rules governing how
tran-
scription factors that direct cellular lineages act informs
directed
attempts to reprogram one lineage to another. In an early
exam-
ple based on such logic, Querfurth et al. (2000) queried the
sig-
nificance of the lack of FOG-1 in eosinophils, despite their
de-
pendence on GATA-1 for differentiation. Forced expression of
FOG-1 in avian eosinophils downregulated expression and
func-
tion of C/EBPb, an essential eosinophil factor, leading to
acqui-
sition of a multipotent phenotype. Conversely, downregulation
of
FOG-1 by C/EBPb is a critical step in eosinophil lineage
commit-
ment. In other experiments, forced expression of GATA-1 was
shown to drive early myeloid avian progenitors to erythroid,
eosinophilic, or thromboblastic (megakaryocytic) precursors
(Kulessa et al., 1995). Moreover, introduction of GATA-1
into
GMPs and common lymphoid progenitors redirects their com-
mitment to megakaryocytic/erythroid progenitors or to
erythroid
cells/mast cells/basophils (Iwasaki and Akashi, 2007).
Commit-
ted B- and T-lymphoid cells can be reprogrammed to
functional
macrophages through expression of C/EBPa (Laiosa et al.,
2006;
Xie et al., 2004). Furthermore, preT cells can be
reprogrammed
to myeloid dendritic cells through PU.1 expression. Cells
that
are reprogrammed transit through an intermediate state in
which
markers of both myeloid and lymphoid lineages are expressed,
indicative of the stepwise nature of the process. Resolution
of
the intermediate state leads to stable unilineage
differentiation.
Notch and GATA-3 appear to counteract reprogramming of
preT cells to macrophages or dendritic cells. Recently,
GATA-
3, traditionally viewed as a T cell-restricted factor, has
been
shown to direct mast cell reprogramming from proT cells (Ta-
ghon et al., 2007). In addition, mid-stage thymocytes (at
the
DN2 stage) can be converted to mast cells through growth in
cul-
ture medium containing interleukin (IL)-3 and stem cell
factor
(Kit-ligand) in the absence of Notch.
Cancer: A Perturbation of the Hematopoietic
Transcriptional Network
Of the more than two dozen regulators designated
‘‘hematopoi-
etic transcription factors,’’ nearly all are intimately
associated
with hematopoietic malignancy (Figure 4). Indeed, the
majority
of genes encoding these factors were discovered either
through
analysis of chromosomal translocations found in human leuke-
mias or study of cooperating leukemia genes during
insertional
mutagenesis in the mouse. Disturbance of the homeostatic
bal-
ance of the critical transcriptional regulators is a defining
feature
of leukemias. In the setting of chromosomal translocations
that
lead to misexpression of a factor, such as SCL/tal1 or LMO2,
tar-
get genes may be inappropriately activated or repressed in
early
lymphoid progenitors. Chimeric transcription factors
generated
through chromosomal translocations exert multiple downstream
effects, including improper target gene activation or
repression,
Figure 6. Reprogramming of Hematopoietic Lineages
The orange arrows depict lineage reprogramming upon expression
of the tran-
scription factors GATA-1, C/EBP, or GATA-3. Abbreviations: HSC,
hemato-
poietic stem cell; CMP, common myeloid progenitor; CLP, common
lymphoid
progenitor; MEP, megakaryocyte/erythroid progenitor; GMP,
granulocyte/
macrophage progenitor.
640 Cell 132, 631–644, February 22, 2008 ª2008 Elsevier Inc.
-
inhibition of function of other critical factors, and
recruitment of
alternative chromatin-modifying enzymes to target loci
(Rose-
nbauer and Tenen, 2007). Leukemia is not the consequence of
nonspecific transcriptional effects but rather the end result of
at-
tacks at vulnerable points in a network. This observation is
best
exemplified by somatic mutations in GATA-1 in Down Syn-
drome-associated megakaryocytic leukemia (Wechsler et al.,
2002), PU.1 and C/EBPa in myeloid leukemias (Mueller et al.,
2002; Pabst et al., 2001), and Pax5 and other B cell factors
(for
example, E2A and EBF) in B-lymphoid leukemias (Mullighan
et al., 2007). In addition to somatic mutation of the principal
he-
matopoietic transcription factors, lesions in more broadly
utilized
signaling pathways that control specific lineage
differentiation
may underlie hematopoietic malignancy. This circumstance is
il-
lustrated by consistent somatic mutation of Notch in T cell
leuke-
mias (Weng et al., 2004). As an essential component of the
reg-
ulatory network for T cell commitment and development, Notch
is a preferred target for somatic mutation in T cell
leukemia.
Messages for the Wider Stem Cell FieldAs arguably the most
‘‘mature’’ organ system under study, the
hematopoietic system constitutes a model for other subfields
of stem cell biology. The hematopoietic system continues to
evolve as a model, however, as numerous critical issues
remain
to be addressed. It is likely that many of the concepts
derived
from work in the blood field will be revisited in other organ
sys-
tems. Nonetheless, it is also apparent that nature has
explored
alternative pathways to tissue organization and development.
Hence, differences should be anticipated, particularly with
re-
spect to the extent to which true stem cells are required for
the
maintenance of different tissues or cell populations within an
or-
gan. Adult stem cell populations are generated during
embryo-
genesis. For other organs, distinct stage-specific programs
reg-
ulate stem cell homeostasis and tissue differentiation. As
illustrated by umbilical cord blood stem cells, transient
popula-
tions may have therapeutic value. Reflection on the history
and
recent advances in the hematopoietic field leads to several
‘‘les-
sons’’ for the stem cell field:
� Precise characterization of the cells within the
hematopoietichierarchy has been instrumental in providing an
adequate frame-
work for biological and molecular studies. The prospective
isola-
tion of subsets of cells, coupled with in vitro
colony-forming
assays and quantitative in vivo transplantation methods, has
greatly facilitated molecular studies of both normal and
malig-
nant hematopoiesis. HSCs differ in their properties
depending
on their location (fetal liver, bone marrow, placenta) and on
the
age of the organism. Hence, thorough cell biological studies
are fundamental to approaching mechanisms that regulate
stem cell function.
� The ‘‘classical’’ hierarchy diagram depicting
progenitorsarising in an orderly fashion from a prototypical HSC
provides
a seductive, but overly simplified view. HSCs may be
described
more accurately as groups of cells with varying
developmental
potentials based on intrinsic networks driven by
transcription
factors and inputs from the cellular niches in which they
reside.
Processes, such as lineage priming coupled with plastic
deci-
sions driven by transcription factor competition, confer
great
flexibility on the options of HSCs. The molecular events of
self-
renewal must be coordinated with these steps. Current views
of HSCs must account for a spectrum of cells with varying
en-
graftment potential and distinct biases toward subsequent
mye-
loid or lymphoid lineage choices. These complex features of
HSC biology are likely to be revisited in studies of other
tissue-
dedicated stem cells.
� Parallel investigation in diverse species has accelerated
anunderstanding of blood cell development. Although an
important
goal is application of fundamental knowledge of
hematopoietic
stem cell biology to the treatment of human disease, studies
in
mice, fish, and flies are mutually reinforcing. Although the
ana-
tomical features of blood formation in the latter organisms
differ
from that of mammals, critical growth factor signaling and
tran-
scriptional pathways are shared. Parallel investigation of
differ-
ent species takes advantage of the strengths of each and
com-
plements work with human hematopoietic cells.
� Elucidation of the transcriptional network that controls
line-age choice and differentiation sets the stage for directed
reprog-
ramming of cellular lineages. Knowing the principal factors
that
govern the cellular lineages identifies candidate regulators
for
functional testing. Further study of the sequence of
molecular
events accompanying lineage conversion is likely to provide
im-
portant mechanistic clues regarding how complex cellular re-
programming occurs (Rossant, 2007).
� The remarkable link between hematopoietic transcriptionfactors
and malignancy underscores how disturbance of a tran-
scriptional network lies at the crux of oncogenesis. As most
he-
matopoietic transcription factors were discovered through
study
of chromosomal translocations in leukemia, investigators
should
pursue the normal developmental functions of genes disrupted
or brought into fusion products in various solid tumors, both
in
sarcomas and epithelial cancers, given the recent
appreciation
for the importance of translocations in these settings. More
gen-
erally, the transcriptional regulators participating in
cell-specific
gene expression provide entry points into the transcriptional
net-
work controlling self-renewal and differentiation, the hallmarks
of
stem cells.
ACKNOWLEDGMENTS
We thank Steve Moskowitz for help with the figures.
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